Inactivation of voltage-gated Na channels is a critically important determinant of the ability of an excitable membrane to transmit an action potential. Fast-inactivation of Na channels occurs over a time course of milliseconds and in part limits the duration of the action potential. A more prolonged form of inactivation, so-called slow inactivation, takes place over several hundred milliseconds and may serve to regulate the excitability of the membrane by limiting the number of Na channels available to participate in the upstroke of an action potential. Recent studies have implicated derangements of slow inactivation in human diseases such as Hyperkalemic Periodic Paralysis. Whereas the structures that participate in fast inactivation are well described, the nature and location of the slow inactivation gating mechanism remains poorly understood. Better understanding of these structures may provide a basis for more rational therapies for disorders of membrane excitability. In our initial proposal we outlined our plan to define those regions of the Na channel that participate in slow inactivation, by examining both regions that may act as the pore-occluding "gate," as well as those structures which may confer voltage sensitivity to slow inactivation. Since our submission, several lines of evidence have emerged and led to the as-yet untested proposal that a collapse near the external face of the ion-conducting pore impedes Na current during slow inactivation. We have tested this hypothesis and have generated evidence that the outer mouth of the Na permeation pathway remains open while the channel is slow inactivated. [This resubmission for a two-year extension has three Specific Aims: 1) To test the hypothesis that some or all of the S4 voltage sensors are immobilized in the outward position during slow inactivation. (2) To test the hypothesis that slow inactivation gating involves conformational shifts in the cytoplasmic portion of the pore, rather than the external pore region as previously hypothesized. 3) To characterize slow inactivation gating of wild-type and disease-associated mutant channels during trains of brief repetitive depolarizations, which will provide greater insight on the physiological relevance of slow inactivation. The long-range career plan of the applicant is to combine the electrophysiological approaches of the on-going work with his former training in cellular and molecular biology to investigate disorders of excitability in neurological disease. The additional two years of training are required to make the transition from being merely acquainted with the techniques of electrophysiology to having the experience to set-up and direct a research program for which electrophysiological techniques are an integral component.]